Denaturation

Denaturation, partial change primarily in the external properties of a substance, not associated with substantial changes in its chemical composition or structure. By denaturation of proteins is usually meant such changes that are primarily reflected in their colloidal properties, particularly in the solubility of the protein, while the chemical structure of its molecule does not essentially change. As a result of denaturation, the protein, which in its natural state (native protein) is a lyophilic colloid, acquires the properties of a lyophobic colloid and under the influence of various conditions easily precipitates from solution, coagulates. Coagulation, indeed, and moreover irreversible, is usually the most obvious manifestation of denaturation. A number of names have been proposed to denote denatured proteins (alloproteins, metaproteins, proteans of English and American authors, protalbuminic and lysalbuminic acid, etc.); they have not received general acceptance. Among the factors causing denaturation of protein, the greatest importance and best studied is the effect of heating. Nothing definite is yet known about the nature of the chemical changes that occur in the protein molecule during this process, nor with respect to other types of denaturation. The opinions of individual authors differ even as to whether thermal denaturation is accompanied by the addition of water or, conversely, by dehydration. The fact that the process proceeds according to the type of monomolecular reactions indicates that some chemical changes must occur in it. Externally, thermal denaturation, if a not too concentrated solution of neutral protein is heated, manifests itself in the turbidity of the solution and, in the presence of sufficient amounts of neutral salts, in the precipitation of the denatured protein in the form of flakes. In the absence of salts, precipitation may not occur, but it happens upon their subsequent addition. If heating is carried out under alkaline or strongly acidic conditions, then albuminates are formed (see). A characteristic property of denatured protein is that it completely precipitates at the isoelectric point. The position of the latter may differ somewhat from the isoelectric point of the native protein. Thus, for native serum albumin, it lies at pH=4.8, and for denatured protein at pH=5.05. In the presence of neutral salts, the isoelectric zone is significantly expanded. In acids and alkalis, denatured protein dissolves as easily as native protein. The temperature required for denaturation of individual proteins varies greatly: it is lowest for tissue proteins, myosin, and fibrinogen; higher for less differentiated albumin and globulin. Previous attempts to distinguish proteins by the temperature of their denaturation were not successful, since not only temperature but also the duration of heating, the reaction of the solution (denaturation is accelerated under strongly acidic or alkaline reaction), salt content, etc., are important. In addition to heating, denaturation of proteins, as far as can be judged by the irreversible loss of solubility, also occurs under the influence of a number of other factors. Even simple storage of proteins in dry form is accompanied by denaturation, especially in globulins, fibrinogen, and myosin. More resistant is crystalline albumin (withstands heating to 120-150° in completely dry form). However, with prolonged storage of crystals in ammonium sulfate solution, they also pass into an insoluble modification, i.e., denature. Partial denaturation apparently also occurs under the influence of conditions existing in the surface layer of solutions. Thus, with vigorous shaking, protein particles accumulating as a result of adsorption at the boundary between the solution and foam bubbles form a kind of film and then lose their solubility. This may explain the inactivation of some enzymes upon shaking. All methods of protein precipitation, except salting out, are accompanied by more or less rapidly occurring denaturation; in particular, when precipitating with alcohol, the precipitate is soluble at first, but with time loses its solubility; this denaturation occurs much faster in the presence of salts. Acetone acts in the same way. Denaturation (i.e., loss of solubility) and coagulation form the basis of all fixation methods used in histological technique (see). Denaturation in the sense as this term is understood at present is accompanied by a change in only one property of the protein - its solubility, while all its other characteristic signs, in particular color reactions, are preserved during denaturation. An exception is the reaction with the formation of lead sulfide, since the so-called loosely bound sulfur, which causes it, is easily cleaved off during the denaturation process.